The present invention provides water soluble polymeric conjugates of camptothecin possessing enhanced antitumor activity and decreased toxicity with respect to the free drug. 7-Ethyl-10-hydroxy-camptothecin (1a, Z=H), a topoisomerase I inhibitor belonging to the class of camptothecin, is a non-soluble compound and it is recognized as the active metabolite of irinotecan (CPT-11, 1b, Z=1,4xe2x80x2-bipiperidinecarbonyl, Cancer Res.: 50,1715-20, 1990). 
The invention provides a polymeric conjugate which is denoted herein a (A) and which consists essentially of:
(i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented by formula (2) 
(ii) from 3 to 15 mol % of units represented by formula (3) 
xe2x80x83wherein [S] is a spacer group; Z is hydrogen, R1CO in which R1 is C1-C6 alkyl or the group 1,4xe2x80x2-bipiperidine and
(iii) from 0 to 12 mol % of -methacryloyl-glycine or N-(2-hydroxypropyl)methacryloyl-glycinamide units represented by formula (4) 
xe2x80x83wherein [X] represents hydroxy or a residue of formula xe2x80x94NHxe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH3.
The polymer of the present invention may also be represented as follows:
[(2)]x; [(3)]y; [(4)]z wherein (2), (3) and (4) are units of the formula as above defined, and x is from 85 to 97 mol %, y is from 3 to 15 mol % and z is from 0 to 12 mol %. Preferably, the polymeric conjugate (A) contains the N-(2-hydroxypropyl) methacryloyl amide units represented by the formula (2) in a proportion of 90% or more; more preferably 90%. The conjugate may also contain from 3 to 10 mol % of the 20-O-(methacryloyl-glycyl-peptidyl)camptothecin units represented by the formula (3), more preferably 10 mol % of such units. Preferably A does not contain residues of formula (4), i.e. z is 0.
The spacer group is susceptible to intratumoral enzymatic hydrolysis. The spacer group may be an amino acid residue or a peptide spacer, for example from 2 to 4 amino acid residues long. Preferably the spacer group [S] is selected from Ala-Gly, Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and Phe-Leu-Gly-Gly.
Preferably, the polymeric conjugate (A) contains the units represented by formula (3) wherein Z is H.
Content of active camptothecin derivative of formula (1) in the conjugate of formula A may be from 2 to 15% (weight/weight), more preferably 10% (w/w).
The invention also provides a process for preparing a polymeric conjugate as defined above, which process comprises reacting a camptothecin derivative of formula (5): 
wherein [S] and Z are as defined above, with a polymer (B) consisting essentially of:
(i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented by formula (2) 
(iv) from 3 to 15 mol % of N-methacryloyl-glycyl units represented by formula (6) 
xe2x80x83wherein R2 is:
the residue of an active ester, or
hydroxy;
and optionally displacing the remaining active ester groups with 1-amino-2-propanol.
The invention also provides 20-O-peptidyl-camptothecin derivatives of formula (5xe2x80x2) 
wherein the group [Sxe2x80x2] is a peptide spacer, for example from 2 to 4 amino acid residues long, preferably selected from Ala-Gly, Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and Phe-Leu-Gly-Gly; Z is as defined above, and their salt derivatives. Such compounds are prepared by condensing camptothecin derivatives of formula 1 as above defined with a N-protected-peptidyl derivative of formula (7):
R3xe2x80x94[Sxe2x80x2]xe2x80x94Pxe2x80x83xe2x80x83(7)
wherein [Sxe2x80x2] is as defined above and R3 represents an amino-protecting group, such as Boc, FMOC, triphenylsilyl, diphenylmethylene or triphenylmethyl, and P is a residue of an activated ester, such as p-nitrophenoxy or N-hydroxysuccinimido, to give a compound represented by formula (8): 
wherein Zxe2x80x2 is R3[Sxe2x80x2] or R1CO, wherein R1, R3 and [Sxe2x80x2] are as defined above and then deblocking the N-protecting group of the substituent at position C-20 from the resulting compound. The compounds of formula (5) as defined above may be analogously prepared.
Preparation of compounds of formula (7) follows standard synthetic procedures that are known from the literature. Suitable N-protected-peptidyl derivatives of formula 7 include, for example, N-(tri-phenylmethyl)-L-phenylalanyl-L-leucyl-glycyl p-nitrophenylester (7a), N-(t-Butoxycarbonyl)-L-phenyl-alanyl-L-leucyl-glycyl p-nitrophenylester (7b). Thus, for example, 7-ethyl-10-hydroxy-camptothecin (1a) may be allowed to react with a molar excess, for example up to five-fold molar excess or more, especially 2 mol.equivalent, of a N-protected-peptidyl derivative of formula (7) in anhydrous solvent such as dry dimethylformamide in the presence of 4-dimethylaminopyridine. In this manner, the protected amino acid is introduced at both hydroxylated positions 10-OH and 20-O of compound 1a to give compound of formula (8a: Z=R3 [Sxe2x80x2]).
The reaction can typically be effected for from 8 to 48 hours. The reaction is typically carried out at a temperature from 15 to 40xc2x0 C. The substituent group at position 10-OH and the amino-protected group R3 of the substituent at 20-OH are removed by an appropriate deprotecting agent to give the 7-ethyl-10-hydroxy-20-O-peptidylcamptothecin derivative of formula (5a: Z=H). Deprotection may be therefore achieved by acid treatment, such as treatment with acetic acid, a mixture of acetic acid and 1.5N aqueous hydrochloric acid or 90% aqueous trifluoroacetic acid from one to 6 hours at temperature from 10 to 30xc2x0 C.; preferably for two hours at room temperature.
The condensation of derivative of formula (5) with the polymer of formula (B), is carried out in conditions capable of preserving the nature of linkage between 7-ethyl-10-(substituted)-camptothecin and spacer [S] as well as that of the conjugate.
Polymers consisting essentially of from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units of formula (2) and from 15 to 3 mol % of N-methacryloyl-glycine units of formula (6), are prepared by copolymerization of N-(2-hydroxypropyl)methacrylamide with N-methacryloyl-glycine or methacryloyl-glycine active-ester derivatives, as described in Makromol.Chem. 178, 2159 (1977). R2 may represent a phenoxy group which is substituted on the phenyl ring by one ore more electron-withdrawing groups, such as nitro or halogen. Preferably R2 represent p-nitrophenoxy. Preferably, the reaction between a polymer in which R2 represents the residue of active ester and a compound of formula (5) to prepare the polymeric conjugate of the invention is carried out in an anhydrous polar organic solvent such as dimethylsulfoxide. The reaction can typically carried out at temperature from 15 to 30xc2x0 C., preferably at room temperature for 15 hours; then the aminolysis of the remaining active ester groups can be performed in the presence of 1-amino-2-propanol at room temperature, from 0.5 to 1 hour. The conjugate suitably is precipitate with ethyl acetate, dissolved in ethanol and reprecipitated with ethyl acetate.
For example, the polymer in which R2 represents the residue of an active ester, provided at a concentration of 15% (w/v) in dry dimethylsulfoxide, is treated with 7-ethyl-10-hydroxy-20-O-peptidyl-camptothecin derivative of formula (5a), 3% (w/v), at room temperature for 15 hours. Then 1-amino-2-propanol, 0.1% (w/v) is added and the reaction mixture is kept at room temperature for 1 hour. The conjugate can be precipitated with ethyl acetate, collected, washed with ethyl acetate, then dissolved with absolute ethanol at a concentration of 10% (w/v) and precipitated again with ethyl acetate to give the conjugate of formula (A) according to the invention.
The content of active drug (1), in polymeric conjugate of the invention is determined by HPLC or absorbance spectroscopy analysis.
The polymer-bound derivatives of formula (A) are in the range of 5.000 to 45.000 molecular weight, preferably from 18.000 to 35.000. Polymeric drug derivatives of formula (A) are water soluble and show enhanced antitumor activity and reduced toxicity in comparison with the free 7-ethyl-10-hydroxy-camptothecin. They are useful in the treatment of leukemia and solid tumors, such as colon, colo-rectal, ovarian, mammary, prostate, lung, kidney and also melanoma tumors. A human can therefore be treated by a method comprising administering thereto a therapeutically effective amount of a polymeric conjugate of the invention. The condition of the human patient can thus be improved.
The dosage range adopted will depend on the route of administration and on the age, weight and condition of the patient being treated. The polymeric conjugates of formula (A) as well as soluble salt derivatives of formula (5xe2x80x2) are typically administered by parenteral route, for example intramuscularly, intravenously or by bolus infusion. A suitable dose range is from 1 to 1000 mg of equivalent per m2 body surface area of active drug of formula (1), for instance from 10 to 100 mg/m2 
The polymeric conjugate (A) or soluble salt derivatives of formula (5xe2x80x2) may be formulated into a pharmaceutical composition together with a pharmaceutically carrier or diluent. Typically they are formulated for parenteral administration, for example by dissolution in water for injection or physiological saline.